Cereulide is an emetic toxin produced by some strains of . This bacterial toxin, a cyclic 1.2 kDa dodecadepsipeptide, is stable to heat and acids and causes nausea and vomiting when ingested via contaminated food. This work aimed to develop and validate a targeted analytical method applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify this toxin in food and human faeces. Samples were extracted with acetonitrile in the presence of C-cereulide, a labelled internal standard, and purified by centrifugation and filtration. The limits of quantification were 0.5 and 0.3 µg kg for food and faeces, respectively. The linearity of the method was very good, with calculated R values above 0.995. The mean recovery of the method was within the acceptable range of 70.0%-120.0%, the repeatability was not higher than 7.3%, and the highest intra-laboratory reproducibility was 8.9%. The estimated range for the expanded measurement uncertainty was between 5.1% and 18.0%. The LC-MS/MS method was used to analyse one food sample (rice) from a Belgian foodborne outbreak and five faecal samples from patients with clinical symptoms after consumption of the contaminated rice. The levels of cereulide were 12.22 µg g for food and between 6.32 and 773.37 ng g for faecal samples.